The present invention relates to the treatment of disseminated fungal infections in mammals. More particularly, it relates to the use of Amphotericin B, encapsulated within liposomes comprised of lipids other than sterols, as a therapeutic agent for the treatment of disseminated fungal infections in mammals.
Infection is the cause of death in 51% of patients with lymphoma and 75% of patients with leukemia. Although bacteria are the causative organisms of many such infections, fungi account for 13% of the fatal infections in patients with lymphoma and for more than 20% of patients with leukemia. The fungus Candida albicans causes more than 80% of these infections, and Aspergillus spp. is also a frequent cause of such infections. In addition, fungal infection is a major cause of morbidity and mortality in patients with congenital and acquired deficiencies of the immune system.
Despite the fact that several new antifungal agents have become available, Amphotericin B remains the drug of choice for treatment of most systemic mycoses in cancer and other immuno-compromised patients. Amphotericin B, a polyene antibiotic, is a lipophilic compound which interacts with ergosterol in fungal membranes, thus creating transmembrane channels which permit the escape of many ions and metabolites that are essential to the cell's continued vitality. Unfortunately, the drug also interacts appreciably with the cholesterol found in mammalian cell membranes. This interaction with the cell membrane of mammalian cells is probably the basis of the toxic effects which it exerts on the mammalian kidney, hematopoietic system and central nervous system.
Amphotericin B is insoluble in aqueous solution, consequently it is supplied commercially as a combination of Amphotericin B, desoxycholate and buffers, suspended in a glucose solution to form a colloidal suspension for administration to the patient. It is usually given intravenously over a period of from two to six hours. Faster infusions may result in cardiotoxicity. Other toxic effects of Amphotericin B may manifest themselves as renal disfunction, anemia, fever and hypotension. Amphotericin B is supplied commercially under the brandname FUNGIZONE.RTM. by E. R. Squibb & Sons, Inc. The side effects and contraindications of FUNGIZONE.RTM. are discussed at page 1929 et seq. of the Physicians' Desk Reference, 37th Ed. (Oradell, N.J., Medical Economics Co., 1983), hereby incorporated by reference.
The toxicity of Amphotericin B limits the total amount of the drug which may be used in the treatment of a fungal infection. Furthermore, it is often ineffective in neutropenic and immunodeficient patients, patients who are highly susceptible to fungal infections. Consequently, there is a need for a system which decreases the toxicity of Amphotericin B to the mammalian system while simultaneously enhancing its effectiveness against the fungal infection.
It has recently been shown that the encapsulation of certain drugs in liposomes before administration to the patient can markedly alter the pharmacokinetics, tissue distribution, metabolism and therapeutic efficacy of these compounds. ("Liposomes" can be defined as lipid vesicles which are formed spontaneously on addition of an aqueous solution to a dry lipid film.) Further, the distribution and pharmacokinetics of these drugs can be modified by altering the lipid composition, size, charge and membrane fluidity of the liposome in which they are encapsulated.
Recently, liposomes have been used as carriers of Amphotericin B for treatment of murine leishmaniasis (New, R. R. C., et al., "Antileishmanial Activity of Amphotericin and Other Antifungal Agents Entrapped in Liposomes." J. Antimicrob. Chemother., vol. 8 (1981), pp. 371-381), histoplasmosis (Taylor, R. L., et al., "Amphotericin B in Liposomes: A Novel Therapy for Histoplasmosis." Am. Rev. Respir. Dis., vol. 125 (1982), pp. 610-611), cryptococosis (Graybill, J. R., et al., "Treatment of Murine Cryptococosis with Liposome-Associated Amphotericin B." J. Infect. Dis., vol. 145 (1982), pp. 748-752), and candidiasis (Tremblay, C., et al., "Comparative Efficacy of Amphotericin B (AMB) and Liposomal AMB (lip-AMB) in Systemic Candidiasis in Mice." Abstr. 1983 ICAAC. No. 755 (1983), p. 222). Liposome-encapsulated Amphotericin B has also been used for treatment of coccidioidomycosis in the Japanese macague (Graybill, J. R., et al., "Treatment of Coccidioidomycosis (coccy) in Primates Using Liposome Associated Amphotericin B (Lipo-AMB)." Abstr. 1982 ICCAC, No. 492 (1982), p. 152).
Administration of Amphotericin B in liposomes provides a more effective method for the treatment of disseminated fungal infections since more of the drug can be given to the infected mammal because of its reduced toxicity.
However, in all above-listed studies, and in all other reported studies of which Applicants are aware (other than their own) in which the composition of the liposomes is given, the liposomes are composed of several lipids, always including at least one sterol. The inclusion of a sterol in the formulation of the liposome was considered essential to the stability of the liposome containing the Amphotericin B. Because it was considered necessary to include a sterol in the lipids making up the liposome, all previous studies of which Applicants are aware were conducted with different proportions or types of sterols in the liposome. These studies represented attempts to define a formulation for optimal stability of the liposome and, simultaneously, increased therapeutic efficacy of Amphotericin B (see, for instance, Barza, M., et al., "Toxicity and Distribution in Mice and Activity In Vitro of Liposomal Amphotericin B (Lip-AMB)." Abstr. 1983 ICCAC, No. 281 (1983), p. 133 and Tremblay, C., et al., supra).
The present invention was made possible by the discovery (1) that sterols are not required to incorporate Amphotericin B into liposomes; (2) that sterols are not essential to the continued stability of an Amphotericin B containing liposome; and (3) that Amphotericin B in sterol-containing liposomes has much less demonstrable in vitro antifungal activity than does Amphotericin B encapsulated in non-sterol containing liposomes. Further, studies have shown that some of the lipids which have been used in the formulation of these Amphotericin B-containing liposomes are themselves toxic. For instance, Graybill, J. R., et al., conducted an experiment (reported in J. Inf. Dis. 145; 748 (1983)) in which mice infected with Cryptococcus neoformans were treated with liposome-associated Amphotericin B. The liposomes used by Graybill were composed of three complex lipids, sphingomyelin, stearylamine and dicetyl phosphate, and a sterol, ergosterol. It has now been shown that dicetyl phosphate and particularly stearylamine are toxic in mammalian systems in that they cause damage to the liver. Poste, Biol. Cell 47; 19 (1983).
Consequently, there is a need for a liposome delivery system for Amphotericin B which is stable and yet easily prepared, requires a minimal amount of lipid per unit of Amphotericin B encapsulated, thereby decreasing the potential toxicity of the lipids in the liposome itself, and which enhances the therapeutic efficacy of Amphotericin B. The present invention is directed to liposome-associated Amphotericin B which offers these advantages in that the liposomes are formulated from lipids other than sterols which were selected for their lack of toxicity to mammals and their ability to encapsulate a greater proportionate amount of Amphotericin B than previous sterol-containing liposome formulations.